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Safe sequencing system

a sequencing system and sequencing technology, applied in the field of nucleic acid sequencing, can solve the problem that mass parallel sequencing cannot be used to detect rare variants, and achieve the effect of sensitive and accurate determination of nucleic acid features or sequences

Active Publication Date: 2014-08-14
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes methods for analyzing nucleic acid sequences using unique identifier (UID) sequences. These UID sequences are attached to the ends of DNA fragments and redundantly determined to accurately represent the DNA fragments. The UID sequences are robust and can accurately represent a DNA fragment even if 1% of the members of a family contain the sequence. The methods also involve amplifying the UID sequences and comparing them to reference sequences. The UID sequences have at least 4 different sequences and are complementary to opposite strands of the DNA fragments. The invention provides sensitive and accurate tools for analyzing nucleic acid sequences.

Problems solved by technology

However, massively parallel sequencing cannot generally be used to detect rare variants because of the high error rate associated with the sequencing process.
Some of these errors presumably result from mutations introduced during template preparation, during the pre-amplification steps required for library preparation and during further solid-phase amplification on the instrument itself.
Other errors are due to base mis-incorporation during sequencing and base-calling errors.

Method used

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Examples

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example 1

Endogenous UIDs

[0039]UIDs, sometimes called barcodes or indexes, can be assigned to nucleic acid fragments in many ways. These include the introduction of exogenous sequences through PCR (40, 41) or ligation (42, 43). Even more simply, randomly sheared genomic DNA inherently contains UIDs consisting of the sequences of the two ends of each sheared fragment (FIG. 2 and FIG. 5). Paired-end sequencing of these fragments yields UID-families that can be analyzed as described above. To employ such endogenous UIDs in Safe-SeqS, we used two separate approaches: one designed to evaluate many genes simultaneously and the other designed to evaluate a single gene fragment in depth (FIG. 2 and FIG. 5, respectively).

[0040]For the evaluation of multiple genes, we ligated standard Illumina sequencing adapters to the ends of sheared DNA fragments to produce a standard sequencing library, then captured genes of interest on a solid phase (44). In this experiment, a library made from the DNA of ˜15,000...

example 2

Exogenous UIDs

[0044]Though the results described above show that Safe-SeqS can increase the reliability of massively parallel sequencing, the number of different molecules that can be examined using endogenous UIDs is limited. For fragments sheared to an average size of 150 bp (range 125-175), 36 base paired-end sequencing can evaluate a maximum of ˜7,200 different molecules containing a specific mutation (2 reads×2 orientations×36 bases / read×50 base variation on either end of the fragment). In practice, the actual number of UIDs is smaller because the shearing process is not entirely random.

[0045]To make more efficient use of the original templates, we developed a Safe-SeqS strategy that employed a minimum number of enzymatic steps. This strategy also permitted the use of degraded or damaged DNA, such as found in clinical specimens or after bisulfite-treatment for the examination of cytosine methylation (45). As depicted in FIG. 3, this strategy employs two sets of PCR primers. The...

example 3

Analysis of DNA Polymerase Fidelity

[0046]Measurement of the error rates of DNA polymerases is essential for their characterization and dictates the situations in which these enzymes can be used. We chose to measure the error rate of Phusion polymerase, as this polymerase has one of the lowest reported error frequencies of any commercially available enzyme and therefore poses a particular challenge for an in vitro-based approach. We first amplified a single human DNA template molecule, comprising a segment of an arbitrarily chosen human gene, through 19 rounds of PCR. The PCR products from these amplifications, in their entirety, were used as templates for Safe-SeqS as described in FIG. 3. In seven independent experiments of this type, the number of UID-families identified by sequencing was 624,678±421,274, which is consistent with an amplification efficiency of 92±9.6% per round of PCR.

[0047]The error rate of Phusion polymerase, estimated through cloning of PCR products encoding β-g...

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Abstract

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Description

[0001]This invention was made using support from the National Institutes of Health, grants CA62924, CA43460, and CA57345. Certain rights to the invention are retained by the U.S. government under the terms of the grant.TECHNICAL FIELD OF THE INVENTION[0002]This invention is related to the area of nucleic acid sequencing. In particular, it relates to manipulative and analytic steps for analyzing and verifying the products of low frequency events.BACKGROUND OF THE INVENTION[0003]Genetic mutations underlie many aspects of life and death—through evolution and disease, respectively. Accordingly, their measurement is critical to several fields of research. Luria and Delbrück's classic fluctuation analysis is a prototypic example of the insights into biological processes that can be gained simply by counting the number of mutations in carefully controlled experiments (1). Counting de novo mutations in humans, not present in their parents, have similarly led to new insights into the rate at...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q2563/179C12Q1/6869C12Q1/6874C12Q2535/122C12Q2565/514C12Q1/6806C12Q2521/501C12Q2525/155C12Q2525/179C12Q2525/191C12Q1/6876C12Q2600/158
Inventor VOGELSTEIN, BERTKINZLER, KENNETH W.PAPADOPOULOS, NICKOLASKINDE, ISAAC
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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